巨细胞病毒pp65抗原血症检测

目的：评估巨细胞病毒（CMV）pp65白细胞抗原血症在骨髓移植或外周血干细胞移植后活动性CMV感染早期诊断和CMV 病的预测、指导CMV亚临床期感染的抗病毒治疗及抗病毒疗效判断中的价值。方法：1999年9月－2000年4月共收集 210份乙二胺四乙酸（EDTA）抗凝血，骨髓移植或外周血干细胞移植患者36例。移植前用ELISA法检测患者CMV特异性抗体IgG和IgM，移植后3周起采用CMV Brite试验盒每周检测1次CMV pp65抗原血症，并计数抗原 阳性细胞，直到移植后100d或治疗后抗原阴转、死亡或出院。移植后患者第1次检测到bp65抗原阳性细胞即开始更昔洛韦抗病毒治疗。结果：36例骨髓移植或干细胞移植患者于移植前CMV特异性抗体IgG均阳性，IgM均阴性；移植后16例出现CMV pp65抗原血症，其中15例 出现有症状的CMV感染或CMV病，14例接受更昔洛韦抗病毒治疗，12例治疗后抗原血症阴转、症状消失、死亡2例（病死率14．2％），未经抗病毒治疗者2例均死亡（P＜0．01）。16例pp65抗原阳性患者中，仅2例CMV特异性抗体IgM阳性。10名健康对照组的CMV特异性抗体IgG均阳性，IgM均阴性；pp65抗原血症 检测均阴性。结论：本研究结果提示，人CMV pp65白细胞抗原血症检测可作为骨髓移植后CMV病的预测指标，指导移植后抗病毒治疗的开展和疗效评价。pp65白细胞抗原症出现即开始抗病毒治疗可明显降低病死率。     

人巨细胞病毒 pp65基因重组蛋白在昆虫细胞中的表达与分析

目的为了制备人巨细胞病毒(HCMV)pp65基因疫苗,应用BacToBac系统对HCMVpp65基因进行表达,并对重组蛋白质的特异性及生物活性进行确定。方法通过脂质体法用HCMVpp65基因重组病毒感染Sf9昆虫细胞,并在感染的不同时期收集细胞,用特异性的HCMVpp65MAb对重组蛋白进行Westernblot,并选择HCMVIgG阳性及阴性的孕妇血清与重组蛋白进行免疫印迹反应。结果重组Bacmid大分子DNA直接转染昆虫细胞可得到100%的阳性重组病毒,病毒滴度为3×107pfu/ml,重组病毒组在感染后48h开始出现一相对分子质量为65000大小的特异带,感染后72h量明显增加,持续至感染后96h,在野生型AcNPV感染细胞及正常昆虫细胞的空白对照中未见该蛋白带。Westernblot显示目的蛋白条带与HCMVpp65MAb发生特异反应。6例阳性血清中有5例与重组pp65发生特异性反应,而阴性对照组未见特异性反应带。结论HCMVpp65基因可在Sf9昆虫细胞中有效表达,且所表达的重组蛋白pp65与人体中感染的野生型HCMVpp65具有相同的抗原性。因此有望为疫苗的制备打下基础。     

婴儿人巨细胞病毒感染病原学和临床分析

目的了解婴儿人巨细胞病毒（HCMV）活动性感染的疾病诊断情况，探讨病原学结果与临床症状之间的关系。方法经血清HCMV IgM和抗凝外周血PP65抗原检测确定的HCMV活动性感染的婴儿378例，对其疾病诊断、病原学结果和临床症状之间的关系进行分析。其中107例患儿，使用套式聚合酶链反应（nPCR）加限制性长度多态性分析（RFLP）进行HCMV糖蛋白B（gB）基因分型。结果在378例HCMV感染患儿中，全身性感染和单脏器感染分别占27．78％和72．22％，肝炎、HCMV包涵体病、血小板减少性紫癜、肺炎等4种疾病分别以33．07％、27．78％、13．49％、6．35％占较大比例。≤2周HCMV包涵体病患儿的比例，明显多于3～12周（P〈0．05）和〉12周的患儿（P〈0．01）。高水平PP65阳性细胞率的患儿全身性感染率明显高于低水平PP65阳性细胞率的患儿（P〈0．01）。HCMV IgM和HCMV PP65、HCMV IgM和HCMV DNA、HCMVPP65和HCMV DNA的阳性符合率分别为31．4％、61．4％、57．1％。三项指标均阳性的患儿全身性感染的比例，明显高于一项或两项指标阳性的患儿（P〈0．01）；IgM和HCMV PP65两项阳性的患儿全身性感染的比例，也明显高于其中一项阳性患儿（P〈0．01）。107例患儿HCMV糖蛋白B（gB）基因的分型结果为gBI型53例，gBⅡ型20例，gBⅢ型18例，gBⅠ、Ⅱ混合型7例，gBⅠ、Ⅲ混合型5例，gBⅡ、Ⅲ混合型4例，未发现gBⅣ型。gBⅠ型占多数（49．53％）。结论婴儿HCMV感染临床表现多样；多种指标的检测，可提高检出率；婴儿HCMV感染，以gBⅠ型HCMV占多数。     

尿液HCMV-DNA和血清HCMV-IgM抗体诊断人巨细胞病毒感染的价值

目的提高人巨细胞病毒（HCMV）感染的诊断水平。方法对960例临床疑诊HCMV感染患儿分别应用FQ—PCR、CLIA法检测尿液HCMV—DNA及血清HCMV-IgM抗体水平。结果尿液HCMV-DNA阳性478例,HCMV-IgM抗体阳性206例;二者均阳性191例。478例尿液HCMV—DNA阳性者中,血清IgM抗体阳性HCMV-DNA拷贝数对数均值高于阴性者（P〈0．01）。结论尿液HCMV-DNA定量和血清HCMV-IgM抗体联合检测有助于HCMV感染的诊断。     

人巨细胞病毒截短被膜磷蛋白pp65的原核表达及抗原性分析

目的利用大肠杆菌表达系统表达截短人巨细胞病毒(HCMV)pp65蛋白,并对其抗原性进行初步鉴定。方法采用PCR扩增HCMV pp65基因961～1 686 bp(321aa-562aa)片段,并将其插入表达载体pET30a(+),转化BL21(DE3)菌株,十二烷基硫酸钠—聚丙稀酰胺凝胶(SDS-PAGE)分析蛋白表达,常规镍柱法纯化目的蛋白,以ELISA法初步检测其抗原性。结果获得相对分子质量约为30 kD的融合蛋白,pp65抗原和全病毒抗原的P/N值分别为2.737、2.535。结论本研究成功表达截短HCMV pp65抗原,并证实其蛋白抗原性优于全病毒抗原;此为HCMV检测试剂盒的研制和疫苗的研究提供了依据。     

PCR法诊断幽门螺杆菌感染的评价

目的系统评估聚合酶链反应扩增尿素酶Ａ基因（ＰＣＲ法）对幽门螺杆菌（Ｈｐ）感染的诊断价值．方法采用金标准及ＰＣＲ法检测６９例患者（慢性胃炎５２例、消化性溃疡１２例、胃癌５例）胃粘膜内Ｈｐ．结果金标准诊断有Ｈｐ感染３４例，ＰＣＲ法诊断３５例Ｈｐ阳性；金标准诊断３５例无Ｈｐ感染，ＰＣＲ法诊断３４例Ｈｐ阴性．故ＰＣＲ法敏感度为１００％，特异度为９７１％，粗一致性为９８５％，调整一致性为９８６％，误诊率（假阳性率）为２９％，漏诊率（假阴性率）为０，正确诊断指数（ｒ）为１０，阳性预测值（＋ＰＶ）为９７１％，阴性预测值（ＰＶ）为１００％，阳性似然比（ＬＲ＋）为３５，阴性似然比（ＬＲ）为０．结论ＰＣＲ法对胃粘膜Ｈｐ感染的诊断价值高于细菌培养、组织病理学，尿素酶试验各单项方法．     

器官移植受者人巨细胞病毒糖蛋白H基因型分析

目的分析器官移植受者人巨细胞病毒（HCMV）临床分离株糖蛋白H基因型，为进一步研究其致病性、免疫性以及研制HCMV亚单位疫苗提供依据。方法以巢式聚合酶链反应（nested—PCR）检测97例器官移植受者外周血细胞中的巨细胞病毒gH基因并测序分型。结果有23例移植受者的标本经nPCR检出gH基因阳性，阳性率为23．71％，其中骨髓移植受者阳性率10．31％，肝移植受者阳性率5．15％，肾移植受者阳性率8．25％。在23例HCMV分离株中，20例得到gH基因型结果，其中gH1型10例，占50％，gH2型6例，占30％，gH1、2混合型4例，占20％，呈散在分布（χ2=4．00，P〉0．05）；gH基因型在各移植类型间也呈散在分布（χ2=5．652，P〉0．05）。结论HCMV临床分离株糖蛋白基因分别属于gH基因型1～2型，呈散在分布。     

人巨细胞病毒体外感染巨核系细胞的实验研究

目的探讨人巨细胞病毒(HCMV)对人巨核系细胞的抑制作用及其机制。方法采用体外悬浮培养巨核系细胞技术,观察HCMV AD169株对巨核系祖细胞生长的影响;用聚合酶链反应(PCR)检测HCMV感染巨核系细胞中HCMV即刻早期蛋白(IE)DNA的表达;用流式细胞术检测HCMV感染巨核系细胞上HCMVpp65抗原的表达。结果HCMV AD169株在体外能明显抑制巨核系细胞的增殖。悬浮培养第12天,与对照组相比,HCMV感染组的巨核细胞数减少了82.19%(P<0.05)。HCMV感染巨核系细胞有IE蛋白DNA和pp65抗原的表达,其感染率为(24.5±3.1)%。结论HCMV AD169株在体外能抑制巨核系细胞的增殖,提示HCMV感染引起的血小板减少与其抑制巨核系细胞增殖有关。     

肾移植术后巨细胞病毒病的预防--CMV抗原检测指导的预防性抗病毒治疗

目的 :探讨肾移植术后巨细胞病毒 (CMV)病的预防。　 方法 :采用免疫组化法检测外周血白细胞CMV PP65抗原 ,术后头 3个月每周检查 1次 ,初次检测CMV PP65抗原阳性者 ,2 4h内给予丙氧鸟苷 2 5～ 5mg/(kg·d)静滴 ,疗程至少 4周或至CMV PP65抗原阴转后续用 7～ 14天 ;无效或出现丙氧鸟苷毒副作用时改用磷甲酸钠。　结果 :10 0例受者术后头 3个月检出CMV PP65抗原阳性 4 2例 ,5 8例抗原阴性的受者仅 1例发生CMV病。 4 2例CMV PP65抗原阳性受者丙氧鸟苷治疗后 3 6例 ( 85 7% )抗原阴转 ,6例经磷甲酸钠治疗抗原也阴转 ,平均阴转时间为 2 4 6± 11 3天 ,平均疗程为 3 4 2± 7 1天 ;仅 1例发展为CMV病。与没有给予预防性抗病毒治疗的对照组受者相比 ,CMV病的发病率和死亡率 (P <0 0 1)明显降低。　 结论 :CMV PP65抗原检测可用于指导肾移植受者CMV感染的预防性治疗 ;预防性抗病毒治疗可有效降低CMV抗原阳性者CMV病的发病率和死亡率     

66例亲属活体肾移植的临床观察

目的：探讨人类白细胞抗原（HLA）配型和抗HLA抗体检测在亲属活体肾移植的应用，及其与移植效果的关系。方法：移植受者及其亲属供体采用PCR—SSP进行HLA配型，移植术前行补体依赖微量细胞毒性试验（CDC）交叉配型，同时采用酶联免疫吸附（ELISA）方法筛查和监测受者的群体反应性抗体（PRA）。分析亲属活体供肾移植的临床效果。结果：够例受者接受了亲属活体供肾移植手术。63例术前抗HLA抗体检测为阴性，其中供受者HLA配型错配（MM）者0-3MM受者57例，3例（5．26％）发生肾功能延迟恢复（DGF），2例（3．51％）发生急性排斥（AR）；HLA4～6MM受者6例中分别有1例（16．67％）和2例（33．33％）发生DGF和AR。HLA0～3MM组AR发生率显著性低于4～6MM组（P〈0．05）。而不同临床免疫抑制方案组之间AR发生率无显著性统计学差异（P〉0．05）。甲基泼尼松龙（MP）和抗人胸腺细胞球蛋白（ATG）冲击等治疗措施后，所有AR得以逆转。3例术前预致敏（PRA35％～87．5％）的受者，HLA2～3MM。1例致敏受者肾功能顺利恢复，PRA维持不变。另2例致敏受者术前自然或经血浆置换后PRA转阴，术后发生AR，ATG冲击治疗后肾功能也恢复正常，抗体有轻度反弹。结论：亲属活体供肾移植的组织配型良好，配合抗HLA抗体监测，使得受者移植的机会增加，排斥反应发生率低，但错配者多，术前致敏受者排斥反应发生率高，应加强围手术期处理。     

The immunoneuroendocrine role of melatonin

Abstract: A tight, physiological link between the pineal gland and the immune system is emerging from a series of experimental studies. This link might reflect the evolutionary connection between self-recognition and reproduction. Pinealectomy or other experimental methods which inhibit melatonin synthesis and secretion induce a state of immunodepression which is counteracted by melatonin. In general, melatonin seems to have an immunoenhancing effect that is particularly apparent in immunodepressive states. The negative effect of acute stress or immunosuppressive pharmacological treatments on various immune parameters are counteracted by melatonin. It seems important to note that one of the main targets of melatonin is the thymus, i.e., the central organ of the immune system. The clinical use of melatonin as an immunotherapeutic agent seems promising in primary and secondary immunodeficiencies as well as in cancer immunotherapy. The immunoenhancing action of melatonin seems to be mediated by T-helper cell-derived opioid peptides as well as by lymphokines and, perhaps, by pituitary hormones. Melatonin-induced-immuno-opioids (MHO) and lymphokines imply the presence of specific binding sites or melatonin receptors on cells of the immune system. On the other hand, lymphokines such as -γ-interferon and interleukin-2 as well as thymic hormones can modulate the synthesis of melatonin in the pineal gland. The pineal gland might thus be viewed as the crux of a sophisticated immunoneuroendocrine network which functions as an unconscious, diffuse sensory organ.     

Changes in connexin43, the gap junction protein of astrocytes, during development of the rat pineal gland

Abstract: The abundance of gap junctions between rat pineal astrocytes formed by connexin43 (Cx43) was studied during development. Levels and distribution of Cx43 were measured by immunoblotting and indirect immunofluorescence, respectively. The amount of Cx43 in cells located within the gland was low until about the 7th postnatal day and increased to adult values between the 14th and 21st days postpartum. Although astrocytes, recognized by their vimentin immunoreactivity, were scarce before birth, they were abundant by the 7th postnatal day suggesting that the low levels of Cx43 found at this age corresponded to a low expression of this protein. Localization of the immunoreactivity to Cx43 and vimentin showed a close correlation, indicating that mature or immature pineal astrocytes form gap junctions made of Cx43. Since Cx43 levels attained their adult values at about the time the innervation and the functional state of the gland reached maturity (2–3 weeks after birth), it is proposed that astrocyte gap junctions are involved in the function of the adult rat pineal gland.     

Conservative management of perforated duodenal diverticulum： A case report and review of the literature

Duodenal diverticula are a relatively common condition. They are asymptomatic, unless they become complicated, with perforation being the rarest but most severe complication. Surgical treatment is the most frequently performed approach. We report the case of a patient with a perforated duodenal diverticulum, which was diagnosed early and treated conservatively with antibiotics and percutaneous drainage of secondary retroperitoneal abscesses. We suggest this method could be an acceptable option for the management of similar cases, provided that the patient is in good general condition and without septic signs.     

Response of rat pineal melatonin to calcium, magnesium, and lithium is circadian stage dependent

Abstract: Herein we documented the response of pineal melatonin production to electrolytes known to be effective on pineal function in view of a possible circadian stage dependence. We studied the release of melatonin by perifused rat pineal glands at 2 different circadian stages corresponding to the middle of the light and dark periods, i.e., respectively, 7 and 19 HALO (Hours After Light Onset, L:D = 12:12). The initial efflux rates were, as expected, much higher in the perifusates of glands removed from rats sacrificed during the dark phase than of those removed during the light phase. After 3 hr of perifusion, melatonin release reached similar levels which were found constant up to the 8th hr of perifusion, whatever the circadian stage. Perifusion of the glands with physiological concentrations for the rat of calcium (5.2 mmol/1) and magnesium (1.34 mmol/1) resulted in a stimulatory effect on the pineal glands removed from rats sacrificed in the middle of the dark period (19 HALO), whereas no effects were observed on the pineal glands removed from rats sacrificed during the light (7 HALO). Lithium (0.28 and 0.55 mmol/1) was ineffective on melatonin release in pineal glands removed 7 and 19 HALO. Our results show differences in the initial efflux rates of melatonin and in the response of perifused pineal glands to calcium and magnesium according to the circadian stage.     

Presence of immunoreactive growth hormone and prolactin in the ovine pineal gland

Abstract: The use of antisera raised against bovine growth hormone (GH) and ovine prolactin (PRL) enabled the detection of related immunoreactive (ir) sequences of proteins in ovine pineal tissue. The isolation of PRL-like ir-material was accomplished using a 0.25 M ammonium sulphate (pH 5.5) extraction followed by ethanol precipitation, whereas the resulting 2.0 M ammonium sulphate (pH 7.0) precipitate contained a GH-like immunoreactivity. Gel chromatography of the GH-like immunoreactivity (Sephadex G-100) indicated the presence of several GH-like fragments ranging in the M_r range of 7,000 to 55,000. Analyses of the PRL-like ir-material found in pineal tissue on HPLC using a TSK 545-DEAE column led to the resolution into a single peak of immunoreactivity. A single peak of activity was also observed following chromatofocusing and hydrophobic interaction chromatography of the ir-peak from the TSK 545-DEAE column. The PRL-like ir-material inhibited the binding of [¹²⁵I]ovine PRL-S14 to anti-ovine PRL antibodies without showing an affinity for binding to anti-rat PRL or anti-bovine GH antibodies. Scatchard analysis of the binding of pineal PRL-like ir-material and pituitary ovine PRL-S14 to liver membranes from day-20 pregnant rats revealed similar affinity constants (K_a of 4.7 ± 0.2 × 10⁹ M^-1). In addition, the replication of Nb 2 Node rat lymphoma cells was stimulated by pineal PRL-like ir-material, an effect known to be specific for lactogenic hormones. The pineal PRL-like immunoreactivity appeared on sodium dodecyl sulfate polyacrylamide gels as a single major band of M_r 24,000. The functional status of PRL-and GH-like ir-material in the ovine pineal remains to be determined, but evidence is presented that the overall protein synthesis rate of the rat pineal responded to circulating concentrations of PRL.     

Microbiology of human immunodeficiency virus anorectal disease

PURPOSE: Individuals who are seropositive for the human immunodeficiency virus are at high risk for opportunistic infection and anorectal disorders. Little prospective information is available regarding anorectal pathogens in these patients. METHODS: One hundred sixty-three HIV-seropositive patients presented to the colorectal clinic between 1989 and 1992. Forty-seven (29 percent) patients were thought to have an infectious process and were prospectively studied using a standardized multiculture protocol. RESULTS: Mean age was 33 (range, 19–59) years. All were male; high-risk behavior accounted for 87 percent of HIV transmissions. Presenting complaints included anorectal pain (79 percent), pus per anum (28 percent), and blood per anum (26 percent). Examination revealed perianal tenderness (60 percent), condyloma (38 percent), perianal ulcers (38 percent), and anal fissures (34 percent). Sixty-six sets of cultures were performed; 28 patients had one set, 15 had two sets, and 4 had three sets. Thirty-two of these 47 patients (68 percent) had positive cultures including herpes (50 percent), cytomegalovirus (25 percent),Neisseria gonorrhoeae (16 percent), chlamydia (16 percent), acidfast bacilli (2 percent), and others (9 percent). Six of 32 patients with positive cultures had more than one organism cultured. Sixteen (50 percent) patients with positive cultures were treated medically, 8 (25 percent) were treated surgically and 8 (25 percent) were treated with both modalities. Sixty-one procedures were performed on 17 patients for condylomata. Eighteen patients had 20 procedures for abscesses, 50 percent of whom had positive cultures for other than common bowel flora; all improved. Fourteen patients underwent 33 procedures for perianal fistulas.Mycobacterium fortuitum was cultured from one patient who required 13 procedures for abscesses and fistulas. Forty-five (96 percent) patients were followed for an average of 12.5 months ±2.9 SEM (range, 1–94 months). Symptoms were improved or resolved in 22 of 32 (69 percent) patients with positive cultures and in 11 of 13 (84 percent) with negative cultures. CONCLUSIONS: Specific pathogens may often be identified in human immunodeficiency virus-seropositive patients with anorectal disorders if aggressively sought. Although patients without specific pathogens identified may be expected to improve with planned empiric treatment, positive identification allows more directed therapy.